A thermophilic 8-oxoguanine DNA glycosylase from Thermococcus barophiluss Ch5 is a new member of AGOG DNA glycosylase family

8-Oxoguanine (8oxoG) in DNA is a major oxidized base that poses a severe threat to genome stability. To counteract the mutagenic effect generated by 8oxoG in DNA, cells have evolved 8oxoG DNA glycosylase (OGG) that can excise this oxidized base from DNA. Currently, OGG enzymes have been divided into three families: OGG1, OGG2 and AGOG (archaeal 8oxoG DNA glycosylase). Due to the limited reports, our understanding on AGOG enzymes remains incomplete. Herein, we present evidence that an AGOG from the hyperthermophilic euryarchaeon Thermo Coccus barophilus Ch5 (Tb-AGOG) excises 8oxoG from DNA at high temperature. The enzyme displays maximum efficiency at 75°C–95°C and at pH 9.0. As expected, Tb-AGOG is a bifunctional glycosylase that harbors glycosylase activity and AP (apurinic/apyrimidinic) lyase activity. Importantly, we reveal for the first time that residue D41 in Tb-AGOG is essential for 8oxoG excision and intermediate formation, but not essential for DNA binding or AP cleavage. Furthermore, residue E79 in Tb-AGOG is essential for 8oxoG excision and intermediate formation, and is partially involved in DNA binding and AP cleavage, which has not been described among the reported AGOG members to date. Overall, our work provides new insights into catalytic mechanism of AGOG enzymes.


Introduction
8-Oxoguanine (8oxoG) is one of the major and most deleterious products of oxidized bases in DNA that arises from reactive oxygen species in vivo and oxidizing agents or ionizing radiation in vitro [1]. In addition to forming a canonical Watson-Crick pair with cytosine (8oxoG:C) in the anti-conformation, 8oxoG can form a stable Hoogsteen pair with adenine (8oxoG:A) in the syn-conformation, thus leading to G:C→T:A mutation when dAMP is incorporated opposite 8oxoG during replication [2,3]. Additionally, A:T→C:G mutation can be generated by incorporating 8-oxodGMP that originates from dGTP oxidation opposite a template base dA by DNA polymerase [3]. Thus, 8oxoG in DNA needs to be repaired, since it is mutagenic to cells. Fortunately, cells have evolved a base excision repair process that is triggered by 8oxoG DNA glycosylase (OGG) for repairing 8oxoG in DNA. Additionally, MutY/MutYH and MutT/MTH1 enzymes are important enzymes for such DNA repair system [4].
Sequence analysis demonstrates that OGG enzymes harbor a Helix-hairpin-Helix (HhH) signature motif, belonging to the HhH superfamily [5,6]. The conserved lysine in HhH motif in OGG enzymes is a key catalytic residue for catalysis and covalent intermediate formation [7][8][9][10]. Thus, OGG enzymes are bifunctional glycosylases that possess glycosylase activity and AP (apurinic/ apyrimidinic) lyase activity, capable of excising 8oxoG from DNA to generate an AP site and further cleave the phosphodiester bond of the created AP site [11].
In the present study, we cloned and expressed the Tb-AGOG gene, purified the gene expression product and characterized it biochemically. Our biochemical data demonstrate that Tb-AGOG can excise 8oxoG from DNA at high temperature and is quite thermostable. The enzyme is a bifunctional DNA glycosylase, harboring glycosylase activity and AP lyase activity as observed in other OGG homologues. Importantly, we dissect the roles of five conserved residues D41, E79, K163, Y174 and D229 in Tb-AGOG in excising 8oxoG from DNA, demonstrating that residues D41, E79, K163, and D229 are critical for 8oxoG removal.

Materials and Methods
Cloning, expression, and purification of Tb-AGOG According to the methods described previously [32], we cloned the gene TBCH5v1_0500 encoding the Tb-AGOG protein into the vector pET-30a (+) (Novagen, Darmstadt, Germany). The sequences of the two primers are listed in Table 1. After being verified by sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) pLysS cells (Transgene, Beijing, China) for protein expression.

Homology modeling of Tb-AGOG
Based on the crystal structure of Pa-AGOG (PDB: 1XQP) as a template, the homology modeling of Tb-AGOG was constructed on a SWISS MODEL server (https://swissmodel.expasy.org). By using PyMOL software (Schrodinger, LLC, https://pymol.org), we illustrated the simply-visualized mutation sites in the modeled structure of Tb-AGOG.

Site-directed mutagenesis
By using a site-directed mutagenesis kit (Transgene), we constructed the Tb-AGOG D41A, E79A, K163A, Y174A and D229A mutant plasmids using the vector plasmid harboring the wild-type (WT) gene as a template. These mutagenic primer sequences are summarized in Table 1. After the desired mutations were verified by sequencing, we expressed and purified the Tb-AGOG mutant proteins as described for the WT protein.

DNA glycosylase/lyase activity assays
We synthesized normal and 8oxoG-containing oligonucleotides labeled with Hex at 5′-terminus at Sangon Biotech Company (Shanghai, China). The Hex-labeled oligonucleotide sequences are summarized in Table 1. The Hex-labeled duplexes were prepared as described previously [32].
The standard DNA glycosylase reactions were performed by incubating 100 nM Tb-AGOG with 50 nM 8oxoG-containing ssDNA

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Biochemical and functional characterization of archaeal 8oxoG DNA glycosylase in the 10 μL buffer (20 mM Glycine-NaOH, pH 9.0, 8% glycerol, and 1 mM DTT) at 75 o C for 1 h, unless stated otherwise. The reactions were terminated with the addition of 98% formamide on ice. The reaction products were heated at 95°C for 3 min and chilled rapidly on ice, and loaded onto a denaturing 12% polyacrylamide gel containing 8 M urea for electrophoresis. After the gels were scanned and visualized with a molecular image analyzer (PharosFx System; Bio-Rad, Hercules, USA), the DNA bands in the gels were quantified with ImageQuant software (Molecular Dynamics, Sunnyvale, USA). The detailed reaction conditions were illustrated in each figure legend. All DNA glycosylase assays were repeated three times.

AP lyase activity assays
The AP lyase activity of Tb-AGOG was determined as described previously [38]. Briefly, we prepared the AP-containing ssDNA by incubating 0.1 μL E. coli UDG (0.1 U) (Thermo Scientific, Waltham, USA) with 100 nM uracil-containing ssDNA in the reaction buffer containing 20 mM Tris-HCl, pH 8.0, 8% glycerol and 1 mM DTT at 37 o C for 15 min as described in the manual instruction. Next, we used the AP-containing ssDNA to determine the AP lyase activity of the WT/mutant Tb-AGOG protein. The AP lyase activity assays were performed by incubating 200 nM WT/mutant Tb-AGOG protein with 100 nM AP-containing ssDNA in the reaction buffer (20 mM Glycine-NaOH, pH 9.0, 8% glycerol, and 1 mM DTT) at 75°C for 1 h. The reactions were terminated and the reaction products were treated as described above. All the AP lyase assays were repeated three times.

DNA binding assays
The DNA-binding assays of Tb-AGOG were performed in a buffer containing 20 mM Tris-HCl, pH 8.0, 100 nM Hex-labeled ssDNA, 1 mM DTT, 8% glycerol and 2000 nM WT/mutant Tb-AGOG protein at 25°C for 10 min. The bound DNA was separated by electrophoresis in a 4% native polyacrylamide gel with 0.1×TBE (Trisborate-EDTA) buffer. The gels were visualized with a molecular image analyzer (Bio-Rad) and the DNA bands were quantified with the ImageQuant software. All DNA-binding assays were repeated three times.

DNA trapping assays
DNA trapping assays were performed as described previously [38]. Briefly, 100 nM WT/mutant Tb-AGOG protein was incubated with 200 nM 8oxoG-containing ssDNA in a 10 μL reaction buffer (20 mM Glycine-NaOH, pH 9.0, 8% glycerol, and 1 mM DTT) in the presence of 15 mM NaBH 4 at 75 o C for 1 h. After the reactions were terminated on ice, the reaction products were heated at 100 o C for 3 min, and loaded with 2.5 μL SDS-loading buffer containing 25 mM Tris-HCl, pH 6.8, 5% SDS, and 50% glycerol into a 10% SDS-PAGE. The gel was visualized and the DNA bands in the gel were quantified as described above. All DNA trapping assays were repeated three times.
A complete phylogenetic analysis showed that the OGG enzymes from several archaeal species belong to the OGG2 and AGOG branches ( Figure 1B). Meanwhile, the bacterial OGG enzymes, such as T. maritime OGG and C. acetobutylicum OGG, are clustered into the OGG1 and OGG2 branches, while the eukaryotic OGG homologues are clustered into the OGG1 branch. Intriguingly, the OGG homologue from Candidatus Korarchaeum is in a distinct evolutionary branch that does not belong to any of the three OGG families.
To dissect the biochemical characteristics and roles of the uncharacterized conserved residues of Tb-AGOG, we cloned the enzyme gene, expressed and purified the gene product. As shown in Figure 1C, we successfully expressed the Tb-AGOG gene with the addition of IPTG, and purified the gene expression product with a molecular weight of~30.6 kDa via cell sonication, heat treatment and Nickel-affinity chromatography.

Tb-AGOG can remove 8oxoG from ssDNA and dsDNA
After purifying Tb-AGOG protein, we determined whether it can remove 8oxoG from ssDNA and dsDNA. Using the 8oxoG-containing ssDNA and 8oxoG:C-containing dsDNA as the substrates, we performed DNA cleavage reactions of Tb-AGOG at 65 o C. As shown in Figure 2A, we observed the cleaved product at the tested enzyme concentrations when using 8oxoG-containing ssDNA as the substrate. By contrast, no cleaved product was observed when normal ssDNA was used as the substrate ( Figure 2B). The enzyme displayed similar efficiency in cleaving 8oxoG:C-containing dsDNA compared with that in cleaving 8oxoG-containing ssDNA, ( Figure  2C). Likewise, Tb-AGOG is inactive to cleaving normal dsDNA ( Figure 2D). Overall, Tb-AGOG can excise 8oxoG from ssDNA and dsDNA with similar efficiency at high temperature.

Biochemical characteristics of Tb-AGOG
We employed 8oxoG-containing ssDNA as the substrate to determine the biochemical characteristics of Tb-AGOG protein. First, we investigated the optimal temperature of the enzyme by performing DNA cleavage reactions at various temperatures ranging from 35 o C to 95 o C. As shown in Figure 3A, the 8oxoG-conatining ssDNA substrate was cleaved by Tb-AGOG with varied efficiencies as the reaction temperature was increased from 35 o C to 95 o C. Specifically, Tb-AGOG displayed maximum efficiency at 75 o C-95 o C, demonstrating that the enzyme has the optimal reaction temperature of 75 o C-95 o C. We further found that after 20 min of heating at 100 o C, the heated Tb-AGOG protein only retained 8% cleavage activity ( Figure 3B), suggesting that the enzyme abolishes most of the activity after heat treatment at 100 o C.
Meanwhile, maximum cleavage efficiency of Tb-AGOG was observed at pH 9.0 ( Figure 3C), suggesting that the optimal reaction pH of the enzyme activity is 9.0. Furthermore, we found that Tb-AGOG is independent of divalent metal ions for excising 8oxoG from DNA, since the enzyme exhibits similar cleavage efficiency in

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Biochemical and functional characterization of archaeal 8oxoG DNA glycosylase the presence of 10 mM EDTA or 2 mM of other divalent metal ions ( Figure 3D). Additionally, we observed that NaCl is not required for Tb-AGOG to excise 8oxoG from DNA ( Figure 3E), and the enzyme activity can be inhibited by high concentration of NaCl. Finally, we investigated substrate specificity of Tb-AGOG using 8oxoG-containing dsDNA with four different mispairs as substrates, and demonstrated that the enzyme displays similar efficiency for cleaving 8oxoG:A-, 8oxoG:T-, 8oxoG:C-or 8oxoG:G-containing dsDNA ( Figure 3F).

Structural modeling of Tb-AGOG
Currently, the crystal structures of Pa-AGOG protein and the AGOG from P. furiosus (1XG7, 4PII) have been solved among the reported AGOG homologues [29]. Although the roles of the several conserved residues in Pa-AGOG have been dissected [31], the functions of lots of conserved residues in AGOG homologues remain unclear. To dissect the structural and functional relationship of Tb-AGOG, we constructed its structure homology model using the crystal structure of Pa-AGOG as a template. As shown in Figure 4A, Tb-

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Biochemical and functional characterization of archaeal 8oxoG DNA glycosylase AGOG harbors 13 α-helices, possessing a non-canonical HhH structure as demonstrated in Pa-AGOG [29]. The residue K163 in the non-canonical HhH motif of Tb-AGOG is conserved in other bifunctional glycosylases, suggesting that this residue should be essential for catalysis. Additionally, 8oxoG is encircled with the conserved residues D41 that is located between α2 and α3, E79 in α5, Y174 in α10, and D229 in α13 (Figures 1 and 4). These residues are highly conserved in other AGOG members, which prompted us to propose that they might play important roles in 8oxoG recognition and removal. To investigate the roles of these conserved re-sidues in the enzyme in removing 8oxoG from DNA, we constructed the Tb-AGOG D41A, E79A, K163A, Y174A, and D229A mutants by site-directed mutagenesis. According to the procedures as described for the WT protein, we successfully purified these mutant proteins ( Figure 4B).

Cleavage of 8oxoG-containing ssDNA by the Tb-AGOG mutants
After purifying the Tb-AGOG mutant proteins, we determined their efficiencies for removing 8oxoG from DNA using 8oxoG-containing ssDNA as the substrate. As shown in Figure 5A, we found that the D41A, K163A and D229A substitutions almost completely abolished the cleavage activity of the enzyme. However, the Y174A mutant retained the activity of the WT enzyme, which indicates that the mutation of Y174 to alanine has a marginal effect on the enzyme activity. Additionally, the E79A mutant displayed the reduced cleavage efficiency (15%), compared with the WT protein, suggesting that the substitution of E79 with alanine causes the compromised activity. Thus, these observations suggest that residues D41, K163 and D229 in Tb-AGOG are essential for excising 8oxoG from DNA, residue E79 is partially responsible for catalysis, but residue Y174 is not essential for catalysis.

AP lyase activity of the Tb-AGOG mutants
As observed in other AGOG homologues [31], Tb-AGOG is a bifunctional glycosylase, harboring the concerted AP lyase activity in

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Biochemical and functional characterization of archaeal 8oxoG DNA glycosylase addition to its glycosylase activity, since the cleaved product of the enzyme appears without treatment with hot NaOH solution. Thus, we determined whether the enzyme can act on AP-containing DNA. We used the AP-containing ssDNA created by removal of uracil from ssDNA by E. coli UDG as substrate to perform DNA cleavage reactions of Tb-AGOG at 75 o C. As shown in Figure 5B, we observed 21% cleavage in the absence of Tb-AGOG, demonstrating that the spontaneous cleavage is due to the incubation of 75 o C for 1 h. Compared with the control reaction without the enzyme, the WT protein displayed the increased cleavage percentage (78%), thereby confirming that the enzyme can cleave the AP-containing DNA substrate.
To explore the contributions of these five interested residues of the enzyme to the AP lyase activity, we also performed the APcontaining ssDNA cleavage assays of the Tb-AGOG mutant proteins. Compared with the WT protein, the D41A mutant displayed similar AP cleavage percentage ( Figure 5B), thereby suggesting that the mutation of D41 to alanine has no significant effect on AP lyase activity of the enzyme. By contrast, the E79A, K163A, Y174A and D229A mutants had the reduced AP cleavage activity with various degrees, which indicates that residues E79, K163, Y174 and D229 are involved in cleaving AP site.

DNA binding of the Tb-AGOG mutants
Next, we investigated whether Tb-AGOG can bind with DNA using 8oxoG-containing ssDNA as substrate. As shown in Figure 6A, we found that Tb-AGOG can bind with 8oxoG-containing ssDNA substrate. Additionally, the enzyme displayed similar normal ssDNA binding when ssDNA without 8oxoG was used as the substrate (data not shown).
In addition, we tested the binding efficiencies of the Tb-AGOG mutants using the 8oxoG-containing ssDNA as substrate. Compared with the WT protein, the E79A and Y174A mutants displayed decreased binding with DNA, suggesting that residues E79 and Y174 are responsible for DNA binding. By contrast, the D41A, K163A and D229A mutants exhibited the comparable DNA binding to the WT protein, which suggests that residues D41, K163, and D229 are not essential for DNA binding.

Intermediate formation of the Tb-AGOG mutants
Tb-AGOG possesses the non-canonical HhH motif that contains the conserved lysine (K163) in other HhH-containing proteins. The conserved lysine in S. cerevisiae OGG is essential for forming covalent intermediate [7], which promoted us to investigate whether a covalent intermediate can be formed between Tb-AGOG and 8oxoG-containing DNA. As shown in Figure 6B, the trapped complex between Tb-AGOG and 8oxoG-containing ssDNA was formed, suggesting that Tb-AGOG can form a covalent intermediate with 8oxoG-containing ssDNA.
Next, we examined the intermediate formation efficiencies of the Tb-AGOG mutants by performing the same DNA trapping assays as described for the WT protein. Compared with the WT protein, the D41A, E79A, K163A and D229A mutants could not form the intermediate, which suggests that residues D41, E79, K163 and D229 are essential for intermediate formation. By contrast, the Y174A mutant displayed similar intermediate formation activity to the WT protein, thereby demonstrating that the residue Y174 plays no role in forming covalent intermediates.

Discussion
In the present study, we presented evidence that the thermophilic AGOG from the hyperthermophilic euryarchaeon T. barophilus Ch5 can remove 8oxoG from DNA at high temperature, thus confirming

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Biochemical and functional characterization of archaeal 8oxoG DNA glycosylase that all AGOG homologues harbor conserved function, since they have evolved from a common ancestor. Intriguingly, Tb-AGOG displays several biochemical characteristics distinct from other AGOG homologues. Additionally, we dissected for the first time the roles of five conserved residues in Tb-AGOG in removal of 8oxoG from DNA, which have not been described to date, thus providing new insights into the catalytic mechanism of archaeal AGOG. Hyperthermophilic Archaea thrive at high temperatures above 80°C, thus facing an increased threat to genome stability, since the rates of spontaneous oxidation are accelerated at elevated temperatures [39][40][41], in addition to the increased deamination rates [42]. Surprisingly, despite living in inhospitable high-temperature environments, HA display spontaneous mutation rates similar to E. coli [43,44], thereby suggesting that they are more efficient than E. coli in repairing damaged DNA, even including DNA oxidation. Interestingly, the OGG enzymes from HA are grouped into OGG2 and AGOG families ( Figure 1B). We compared biochemical characteristics of Tb-AGOG with other reported OGG enzymes from Archaea ( Table 2), demonstrating that they have distinct biochemical characteristics. Firstly, the optimal reaction temperature (75 o C-95 o C) of Tb-AGOG is similar to that of Tg-AGOG, but clearly higher than that of Thermoplasma volcanium OGG (Tv-OGG) and A. fulgidus OGG (Af-OGG) [27,45]. Besides, the optimal reaction pH 9.0 of Tb-AGOG is higher than that of Tg-AGOG, Tv-, Mj-and Af-OGG enzymes, respectively [25,27,32,45]. Although these two enzymes possess high amino acid similarity, Tb-AGOG displays thermostabilty distinct from Tg-AGOG. Additionally, 100 mM NaCl is optimal for Af-OGG to remove 8oxoG from DNA [27]. By comparison, NaCl is not required for Tb-AGOG and Tg-AGOG to excise 8oxoG from DNA, and their activities are inhibited by high concentration of NaCl [32]. However, Tb-AGOG is different from Tg-AGOG in NaCl tolerance.
In addition, Tb-AGOG displays similar efficiency for cleaving 8oxoG:N (A, T, C or G) as observed in M. jannaschii OGG (Mj-OGG), which is distinct from the observations for Tg-AGOG, Pa-AGOG, Tv-OGG, and Af-OGG (Table 2). In addition to OGG enzymes, bacteria and eukarya also encode a MutY protein which is capable of excising adenine in 8oxoG:A mismatch that are formed during replication, suggesting that the MutY and OGG enzymes are complementary in their activities as part of the 8oxoG system of the base excision repair pathway [46][47][48]. Like bacteria and eukarya, HA also encodes the bacterial MutY homologues that excise adenine from a 8oxoG:A mismatch, even including T. barophilus Ch5, which indicates that the MutY homologues in HA may be involved in 8oxoG repair. In combination with the role of the MutY homologue in this archaeon, effective cleavage of 8oxoG from a 8oxoG:A mismatch by Tb-AGOG may allow the cells to rapidly repair 8oxoG prior to replication, thus counteracting mispair by DNA polymerase. However, if the 8oxoG in the DNA arises from the oxidation of a G in a G:C basepair (to make 8oxoG:C) followed by mispairing by DNA polymerase (to make 8oxoG:A), then removing the 8oxoG by OGG will be pro-mutagenic (the adenine should be removed by MutY instead). Residue K163 in Tb-AGOG is in the non-canonical HhH motif, which is found in Pa-AGOG [29]. As reported previously, the conserved lysine in bifunctional DNA glycosylases in Pa-AGOG is a catalytic residue, since the substitution of K140 with Q leads to the loss of activity [30]. In this study, we provide data that support this conclusion, since the substitution of K163 with alanine leads to the loss of 8oxoG removal activity and defect in the covalent intermediate formation. By comparison, the K163A mutation reduced AP cleavage (53% relative to the WT protein; Table 3), thereby suggesting that this residue is partially involved in cleaving AP site. Overall, this work improves our understanding of the function of the conserved lysine residue in the AGOG enzymes.
In addition to the conserved lysine in HhH superfamily, the

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Biochemical and functional characterization of archaeal 8oxoG DNA glycosylase conserved residue D218 in α-helix 13 in Pa-AGOG is another catalytic residue, since the mutation of residue D218 to serine leads to the activity loss [31]. Like the residue D218, the residue D229 in Tb-AGOG, which is analogous to the residue D218 in Pa-AGOG, is essential for 8oxoG removal. Importantly, we first revealed the essential role of the conserved residue D229 in the covalent intermediate formation, which has not been described in the D218 in Pa-AGOG.
Residues D41, E79, and Y174 in Tb-AGOG are conserved in AGOG homologues ( Figure 1A). In this study, we explored the roles of the conserved residues D41, E79, and Y174, and demonstrated that the D41A substitution abolished 8oxoG excision and intermediate formation activity, but had no effect on DNA binding and AP cleavage (Table 3). Additionally, the E79A mutant lacks the intermediate formation activity, and displays reduced GO excision, AP cleavage and DNA binding (Table 3). Furthermore, the Y174A mutant has the WT 8oxoG excision activity and intermediate formation activity, with slightly reduced AP cleavage and DNA binding (Table 3). Therefore, our mutational experiments provide new insights into catalytic mechanism of AGOG.
In summary, we present biochemical and functional data of the thermostable AGOG from the hyperthermophilic euryarchaeon T. barophilus Ch5. Tb-AGOG can remove 8oxoG from DNA at high temperature, with maximum efficiency at 75 o C-95 o C and at pH 9.0. In addition, Tb-AGOG is a bifunctional DNA glycosylase that harbors glycosylase activity and AP lyase activity, as observed in other OGG homologues. Importantly, we reveal for the first time that residues D41 and D229 in Tb-AGOG, which are conserved in other AGOG family members, are essential for catalysis, in addition to the conserved residue K163. Residue E79 is partially responsible for 8oxoG removal, DNA binding, AP cleavage and intermediate formation, and residue Y174 is partially involved in DNA binding and AP cleavage. Overall, our work provides insights into the catalytic mechanism of AGOG.

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Biochemical and functional characterization of archaeal 8oxoG DNA glycosylase